Critical point drying of hydrogels in analyte sensors

ABSTRACT

A hydrogel is dried by a critical point drying technique. The hydrogel may include indicator molecules embedded therein and may be part of a hydrogel-based device, such as, for example, an analyte sensor.

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims the benefit of priority to U.S.Provisional Application Ser. No. 61/879,475, filed on Sep. 18, 2013,which is incorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to critical point drying (“CPD”) and, moreparticularly, to CPD of a hydrogel-based device such as, for example andwithout limitation, a sensor platform of an analyte sensor (e.g., anoptical-based or electrochemical analyte sensor) that uses apolymer-indicator system that is based on hydrogels. CPD allows for ahydrogel to be kept in its hydrated state and improves longevity andshelf life of the sensor and further allows the sensor to be implantedinto a patient without re-hydrating the sensor. CPD dehydrates thehydrogel without leaving it vulnerable to cracking or disrupting itsstructure, and thus ensures proper sensor operation after implantation.

BACKGROUND

An exemplary analyte sensor 10 with which aspects of the invention maybe employed is shown in FIG. 1. The sensor may be implantablesubcutaneously or in the intraperitoneal fluid and is configured fordetecting an analyte of interest, such as glucose. The sensor 10includes a sensor body 12, a matrix layer (or indicator) 14 coated overpart or all of the exterior surface of the sensor body 12, withfluorescent indicator molecules 16 distributed throughout the layer, alight source 18, e.g. an LED, that emits light, including light over arange of wavelengths which interact with the indicator molecules(referred to herein simply as “light at a wavelength which interactswith the indicator molecules”), i.e., in the case of afluorescence-based sensor, a wavelength which causes the indicatormolecules 16 to fluoresce, and a photosensitive element 20, e.g. aphotodetector, which, in the case of a fluorescence-based sensor, issensitive to fluorescent light emitted by the indicator molecules 16such that a signal is generated in response thereto that is indicativeof the level of fluorescence of the indicator molecules.

In some embodiments the indicator molecules are contained within thematrix layer 14, which comprises a biocompatible polymer matrix that isprepared according to methods known in the art and coated on the surfaceof the sensor body. Suitable biocompatible matrix materials, which mustbe permeable to the analyte, include hydrogels which, advantageously,can be made selectively permeable—particularly to the analyte—i.e., theyperform a molecular weight cut-off function. The sensor 10 may alsoinclude reflective coatings 32 formed on the ends of the sensor body 12,between the exterior surface of the sensor body and the matrix layer 14,to maximize or enhance the internal reflection of the light and/or lightemitted by fluorescent indicator molecules.

The sensor 10 may be configured to be implantable in a patient and maybe constructed in such a way that no electrical leads extend into or outof the sensor body to supply power to the sensor (e.g., for driving thesource 18) or to transmit signals from the sensor. Thus, the sensor mayinclude a means for receiving power from an external source 40 that iswholly embedded or encapsulated within the sensor body 12 and atransmitter 42 that also is entirely embedded or encapsulated within thesensor body 12.

As still further illustrated in FIG. 1, an optical filter 34 may beprovided on the light-sensitive surface of the photosensitive element(photodetector) 20. This filter prevents or substantially reduces theamount of light generated by the source 18 from impinging on thephotosensitive surface of the photosensitive element 20. At the sametime, the filter allows fluorescent light emitted by fluorescentindicator molecules 16 to pass through it to strike the photosensitiveregion of the detector. In addition, a temperature sensor 64 and anoptional signal amplifier 66 may also be provided. The temperaturesensor 64 measures the locally surrounding temperature of the ambienttissues and the indicator molecule environment and provides thisinformation to the control logic circuit (not shown). The control logiccircuit correlates fluorescence level, for example, with analyteconcentration level, thereby correcting the output signal for variationsaffected by temperature. Amplifier 66 is a relatively simple gaincircuit which amplifies the signal generated by the photodetector 20.

The various components and circuitry of the sensor 10 may be assembledonto a ceramic (e.g., ferrite) substrate 70.

Exemplary sensors are described in U.S. Pat. Nos. 5,517,313; 6,330,464;and 6,400,974, as well as in U.S. patent application Ser. No.13/761,839, the respective disclosures of which are hereby incorporatedby reference in their entireties.

If a sensor is implanted in the body of a living animal, the animal'simmune system may begin to attack the sensor. For instance, if a sensoris implanted in a human, white blood cells attack the sensor as aforeign body, and, in the initial immune system onslaught, neutrophilsare the primary white blood cells attacking the sensor. The defensemechanism of neutrophils includes the release of highly causticsubstances known as reactive oxygen species. The reactive oxygen speciesinclude hydrogen peroxide.

In some non-limiting embodiments, the indicator 14 may be covered by athin layer (e.g., 10 nm) on the outside of the indicator. The thin layermay protect against indicator molecule degradation. The thin layer maybe platinum, and the platinum may be sputtered onto the outside surfaceof the indicator. Platinum rapidly catalyzes the conversion of hydrogenperoxide into water and oxygen, which are harmless to the sensor. Therate of this reaction is much faster than the boronate oxidation; thus,the platinum provides some protection against oxidation by reactiveoxygen species. Although platinum is the catalyst of the conversion ofhydrogen peroxide into water and oxygen in some embodiments, inalternative embodiments, other catalysts of this reaction, such as, forexample, palladium or catalase, may be used for the thin layer insteadof or in addition to platinum.

Under certain circumstances, after a sensor is assembled, it may bewashed and then air dried (e.g., using heat, ambient air, dry gases orgases of a controlled humidity). When heat dried or air/gas dried,however, the hydrogel tends to shrivel like a dry sponge. Thisshriveling can cause a sputtered protective catalyst (such as platinum)to crack as the sensor is drying, which may pose a problem when thesensor is rehydrated and implanted. Cracking of protective catalyst mayleave areas of the sensor vulnerable to attack by the immune system.

In addition to the physical damage (surface morphology) to the indicatoror platinum layer due to shrinkage and cracking that accompanies airdrying, another challenge involves long hydration periods that arerequired prior to implanting an air-dried sensor—especially after aperiod of storage. For air dried sensors, the average hydration period,or time to reach equilibration of in vivo signal, ranges from 30 minutesto 7 days depending on the storage time of the hydrogel (FIG. 2). Thisinconsistent behavior makes it difficult to predict the hydrationprofile of the hydrogel and time to stable baseline signal once insertedinto a patient. Also, longer storage times in the dried state may causeirreversible signal loss that can never be regained due to theinstability of the hydrogel in its unhydrated state (FIG. 2).

In the example shown in FIG. 2, the expected rehydration point of thehydrogel is shown to reach approximately 6 on the normalized signalscale. As can be seen in the plot, the signals of sensors that have beenstored for 4 weeks reach the expected normalized signal level within afew days. Sensors stored for 8 weeks eventually reach the expectednormalized signal level of 6, but it takes 5-6 days to reach that level.On the other hand, the signal level of sensor stored for 4 months ormore never reach the expected normalized signal level.

Thus, as shown in FIG. 2, as the hydrogel is stored for periods greaterthan one month, the hydration of the hydrogel may take days or evenmonths to equilibrate. Storage longer than 4 months does not allow thehydrogel to rehydrate at all.

Thus, a need exists for techniques for improving the longevity (shelflife) and rehydration characteristics of hydrogels employed in analytesensors while minimizing or eliminating the surface morphologyassociated with air drying.

SUMMARY OF THE INVENTION

Critical point drying (“CPD”) preserves the structure of specimen whichcould otherwise be damaged due to surface tension when changing from aliquid state to a gaseous state. In one embodiment, a CPD techniquedehydrates the hydrogel of a hydrogel-based device (e.g., a sensor)without altering the surface or bulk morphology of the gel. The hydrogelis thus less vulnerable to collapsing, rearranging, or disrupting itsstructure. The hydrogel-based device can be dried easily in a criticalpoint dryer since liquid CO₂ used in the CPD process, for example,changes from the liquid phase to the gas phase instantaneously withoutaffecting the sensor gel morphology. An unaffected sensor morphologyensures proper hydrogel-based device operation after implantation.

Critical point drying is extremely useful for dehydrating, whilemaintaining the structure of the hydrogel. At a specific and fixedtemperature, it has been found that the percent modulation, absolutemodulation, and the reflection of the sensors remains at a highpercentage.

CPD produces a gel with substantially unaltered structure/morphology,yields a faster hydration time, and allows a hydrogel-based device to bestored for an extended period (weeks and months) without degradation ofthe indicator molecule(s). In addition CPD is especially useful fordrying without collapsing or deforming the structure of wet, fragilespecimens (e.g., sensor hydrogel).

One aspect of the invention may provide a method of preparing a hydrogelfor a hydrogel-based device. The method may comprise drying the hydrogelby a critical point drying technique.

In some embodiments, the hydrogel-based device comprises a sensorconfigured for sensing the presence of an analyte in contact with thesensor. The sensor may include an indicator comprising the hydrogel andconfigured to emit a detectable signal or a change in a detectablesignal indicating the presence of the analyte.

In some embodiments, the critical point drying technique may comprise:replacing water within the hydrogel with an intermediate fluid that ismiscible with water and CO₂. The critical point drying technique maycomprise replacing the intermediate fluid within the hydrogel with CO₂.The critical point drying technique may comprise manipulatingtemperature and pressure to achieve super critical CO₂. The criticalpoint drying technique may comprise removing the supercritical CO₂ fromthe hydrogel. In some embodiments, the temperature for achieving supercritical CO₂ may be, preferably, about 30-35° C. and the pressure forachieving supercritical CO₂ may be, preferably, about 1000-1200 psi.

In some embodiments, the method may further comprise storing thehydrogel-based device at room temperature after the critical pointdrying step. In some embodiments, the method may further compriseimplanting the hydrogel-based device into a patient after the criticalpoint drying step without pre-hydrating the hydrogel-based device priorto implanting.

Another aspect of the invention may provide a hydrogel dried by acritical point drying technique.

In some embodiments, the critical point drying technique may maintainthe physical properties of the hydrogel. In some embodiments, thecritical point drying technique may extend the shelf life of thehydrogel. In some embodiments, the critical point drying technique maynot change the performance of the hydrogel in one or more of in vitroand in vivo environments.

Still another aspect of the invention may comprise a sensor configuredfor sensing the presence of an analyte in contact with the sensor. Thesensor may comprise an indicator configured to emit a detectable signalor a change in a detectable signal indicating the presence of theanalyte. The indicator may comprise a hydrogel dried by a critical pointdrying technique.

Further variations encompassed within the devices, systems and methodsare described in the detailed description of the invention below.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated herein and form partof the specification, illustrate various, non-limiting embodiments ofthe present invention. In the drawings, like reference numbers indicateidentical or functionally similar elements.

FIG. 1 is a schematic view of an exemplary analyte sensor incorporatingindicator molecules suspended in a hydrogel.

FIG. 2 is a plot of signal strength versus number of days exposed toglucose for different analyte sensors having been air dried and storedfor various periods of time.

FIG. 3 is a pressure versus temperature plot showing different phasesand the critical point for carbon dioxide CO₂.

FIG. 4 is a plot of signal versus time exposed to rehydrating fluid fora number of analyte sensors dried by a critical point drying techniqueand stored for four months or one month.

FIG. 5 is a table showing glucose response data obtained on afluorometer for a number of sensors dried by a critical point dryingtechnique having been stored for four months at different conditions.

FIG. 6 is a flow chart illustrating a critical point drying techniquefor preparing an analyte sensor with a hydrogel indicator in accordancewith one embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all terms of art, notations and otherscientific terms or terminology used herein have the same meaning as iscommonly understood by one of ordinary skill in the art to which thisdisclosure belongs. All patents, applications, published applications,and other publications referred to herein are incorporated by referencein their entirety. If a definition set forth in this section is contraryto or otherwise inconsistent with a definition set forth in the patents,applications, published applications, and other publications that areherein incorporated by reference, the definition set forth in thissection prevails over the definition that is incorporated herein byreference.

As used herein, “a” or “an” means “at least one” or “one or more.”

This description may use relative spatial and/or orientation terms indescribing the position and/or orientation of a component, apparatus,location, feature, or a portion thereof. Unless specifically stated, orotherwise dictated by the context of the description, such terms,including, without limitation, top, bottom, above, below, under, on topof, upper, lower, left of, right of, inside, outside, inner, outer,proximal, distal, in front of, behind, next to, adjacent, between,horizontal, vertical, diagonal, longitudinal, transverse, etc., are usedfor convenience in referring to such component, apparatus, location,feature, or a portion thereof in the drawings and are not intended to belimiting.

Critical point drying of specimens has been used to process samples forscanning electron microscopy imaging (“SEM”). The process is most usefulfor understanding how a specimen looks like in an in vivo environment,such as tissue or muscle, because it allows the sample to remain in thehydrated state when no water or liquid is present, which is necessaryfor samples in an SEM.

The phase diagram (FIG. 3) shows the pressure to temperature rangeswhere solid, liquid and vapor exist. The boundaries between the phasesmeet at a point on the graph called the triple point. Along the boundarybetween the liquid and vapor phases, it is possible to choose aparticular temperature and corresponding pressure in which liquid andvapor can co-exist (i.e., have the same density). This is known as thecritical temperature and pressure.

Critical point drying relies on this physical principle. The water inthe material is replaced with a suitable inert fluid whose criticaltemperature for a realizable pressure is just above ambient. The choiceof fluids is severely limited (for example, the critical point of wateris about 374° C. and 3212 psi, making it an impractical fluid for CPD),and therefore, CO₂ is the fluid that is often used. With CO₂, a criticalpoint of, preferably, approximately 30-35° C. can be achieved at apressure of, preferably, around 1000-1200 psi. Therefore, if the wateris replaced with liquid CO₂ and the temperature is then raised above thecritical temperature, the liquid CO₂ changes to vapor without change ofdensity and therefore without surface tension effects which can distortthe structure of the material. Since liquid CO₂ is not sufficientlymiscible with water, however, it may be useful to use an intermediatefluid which is miscible with both water and liquid CO₂. In oneembodiment of the CPD process, the intermediate fluid used is methanol.In another embodiment, the intermediate fluid is ethanol or acetone.

Thus, CPD involves the rapid transformation of liquid into gas viaconversion of liquid into a supercritical fluid. This process isachieved by bringing the liquid to its critical point (FIG. 3), viamanipulation of the temperature and pressure, which allows the liquid toskip the phase boundaries between the liquid and gas phases andinstantly become gas, leaving the material dry but locked into its“hydrated” state, i.e., without the surface damage and other morphologydue to surface tension that accompanies phase transitions during airdrying.

This procedure is achieved by replacing water with a liquid that is bothmiscible with water and liquid CO₂, such as methanol, ethanol, acetone,or isopropanol, termed the intermediate fluid. In an exemplary,non-limiting implementation, the material is taken through a series ofintermediate fluid exchanges that slowly replace the water with themiscible liquid until the specimen is completely immersed in 100%intermediate fluid. The specimen then goes through a second series offluid exchanges that replace the miscible intermediate fluid with liquidCO₂, and then the temperature and pressure are manipulated in order tocreate supercritical CO₂ that can instantly be converted to gaseous CO₂leaving the “hydrated” structure in place.

Critical point drying allows a hydrogel to be stored in a hydratedstate, which maintains its physical properties, increases its shelflife, and also eliminates the necessity for a pre-hydration step priorto implant into patients. The CPD process locks the hydrogel in thehydrated state by the instantaneous removal of the supercritical CO₂ viapressure and temperature manipulation forcing the liquid into the gasphase and leaving the hydrogel in its hydrated or expanded state.Examples of some hydrogels that may be dried by a critical point dryingtechnique and/or used in analyte sensing include, but are not limitedto, poly(HEMA), poly(vinyl alcohol), poly(ethylene glycol), silicone,acrylamides and copolymers, and natural hydrogel materials (e.g.,agarose, methylcellulose, hyaluronan).

As explained above and illustrated in FIG. 2, upon insertion intosolution, hydrogel-based devices (e.g., sensors) having air-driedhydrogel take long periods of time to reach equilibrium, depending onhow long the hydrogel is stored dry. In one illustrative example, as thehydrogel sits dry, the useful shelf-life may become undeterminable past8 weeks, and at 8 weeks it takes approximately 5 to 6 days for thehydrogel to equilibrate to its pre-dried signal. FIG. 4 shows sensorsthat were processed by CPD techniques and stored for various amounts oftime followed by monitoring rehydration of the critical point driedhydrogel. The baseline signal reaches a plateau for all sensors storedat 1 and 4 months within 20 minutes and the signal variation during theinitial stabilization period is less than 5% of the baseline signal,meaning that the hydrogel is “hydrated” and potentially ready for invivo use immediately.

The fluorescent response to glucose of the CPD hydrogel were monitoredas a function of storage time and showed no loss of those criticalparameters such as baseline fluorescence (I₀), % modulation, absolutemodulation, T90 and reflectance compared to controls which were neverdried (FIG. 5). In these non-limiting examples, the critical point driedsamples (cores) maintained their high performance even when they werestored for 4 months at room temperature. This advantageous ability tooperate at room temperature improves storage and handling of the finaldevice.

There are advantages (for example, such as those described herein) toutilizing the CPD process which allows for the long-term storage (e.g.approximately 6 months) of hydrogel-based sensor platforms as well asthe ability to maintain both the baseline and modulatable signal. Theprocess also allows for the sensor to be implanted without apre-hydration period—typically a minimum of 6 hours in sterile salinesolution—to allow the air-dried hydrogel to reach some form of hydrationprior to implantation.

An exemplary method for employing a CPD technique for drying a hydrogelof an analyte sensor is shown in FIG. 6. In step 50, a hydrogel isapplied to all or part of the surface of a sensor housing. Step 50 mayor may not be the final step in fabricating the sensor. After step 50,when fabrication of the sensor is complete, or the sensor is otherwiseready to be dried, in step 52, water in the hydrogel is replaced with aliquid that is miscible with both water and liquid CO₂, such as anintermediate fluid such as methanol, ethanol, acetone, or isopropanol,in an intermediate fluid exchange step. Step 52 is performed by takingthe material through a series of intermediate fluid exchanges thatslowly replace the water with the miscible liquid (intermediate fluid)until the specimen is completely immersed in 100% intermediate fluid. Instep 54, the hydrogel goes through a second series of fluid exchangesthat replace the miscible intermediate fluid with liquid CO₂. After theintermediate fluid is replaced by the liquid CO₂, in step 56, thetemperature and pressure of the hydrogel are manipulated to createsupercritical CO₂ that can instantly be converted to gaseous CO₂. Instep 58, the gaseous CO₂ is removed from the hydrogel, leaving thehydrated structure of the hydrogel in place.

Embodiments of the present invention have been fully described abovewith reference to the drawing figures. Although the invention has beendescribed based upon these preferred embodiments, it would be apparentto those of skill in the art that certain modifications, variations, andalternative constructions could be made to the described embodimentswithin the spirit and scope of the invention. For example, although someembodiments described above relate to critical point drying of hydrogelsfor use as an analyte indicator of an analyte sensor, the presentinvention is not limited to critical point drying of hydrogels in theanalyte sensor environment. Some alternative embodiments relate tocritical point drying of hydrogels for use in other hydrogel-baseddevices and/or other environments, such as, for example and withoutlimitation, hydrogels for use as scaffolds in tissue engineering,hydrogels for use as sustained-release drug delivery systems, andhydrogels for use in implantable and non-implantable medical devices.

The invention claimed is:
 1. A method of preparing a hydrogel for ahydrogel-based analyte sensor configured for sensing the presence of ananalyte in contact with the analyte sensor, the method comprising:applying the hydrogel to all or part of a surface of a sensor housing ofthe analyte sensor, wherein the hydrogel comprises indicator moleculesthat are configured to emit a detectable signal or a change in adetectable signal indicating the presence of the analyte; applying aprotective catalyst to the hydrogel, wherein applying the protectivecatalyst to the hydrogel comprises sputtering the protective catalyst onthe hydrogel; and drying the hydrogel, which (i) comprises the indicatormolecules, (ii) has been applied to all or part of the surface of thesensor housing, and (iii) to which the protective catalyst has beenapplied, by a critical point drying technique that comprises: replacingwater within the hydrogel with an intermediate fluid that is misciblewith water and CO₂; replacing the intermediate fluid within the hydrogelwith CO₂; manipulating temperature and pressure to achieve supercritical CO₂; and removing the supercritical CO₂ from the hydrogel. 2.The method of claim 1, wherein the temperature for achieving supercritical CO₂ is about 30-35° C., and the pressure for achievingsupercritical CO₂ is about 1000-1200 psi.
 3. The method of claim 1,further comprising storing the hydrogel-based analyte sensor at roomtemperature after the critical point drying step.
 4. The method of claim1, further comprising implanting the critical point dried hydrogel-basedanalyte sensor into a living being without pre-hydrating thehydrogel-based analyte sensor prior to implanting.
 5. The method ofclaim 1, wherein the protective catalyst is platinum.